Single-cell RNA sequencing identifies response of renal lymphatic endothelial cells to acute kidney injury. Single-cell RNA sequencing identifies response of renal lymphatic endothelial cells to acute kidney injury

Background: The inflammatory response to acute kidney injury (AKI) likely dictates future renal health. Lymphatic vessels are responsible for maintaining tissue homeostasis through transport and immunomodulatory roles. Due to the relative sparsity of lymphatic endothelial cells (LECs) in the kidney, past sequencing efforts have not characterized these cells and their response to AKI. Methods: Here we characterized murine renal LEC subpopulations by single-cell RNA sequencing and investigated their changes in cisplatin AKI 72 hours post injury. Data was processed using the Seurat package. We validated our findings by qPCR in LECs isolated from both cisplatin-injured and ischemia reperfusion injury, by immunofluorescence, and confirmation in in vitro human LECs. Results: We have identified renal LECs and their lymphatic vascular roles that have yet to be characterized in previous studies. We report unique gene changes mapped across control and cisplatin injured conditions. Following AKI, renal LECs alter genes involved in endothelial cell apoptosis and vasculogenic processes as well as immunoregulatory signaling and metabolism. Differences between injury models were also identified with renal LECs further demonstrating changed gene expression between cisplatin and ischemia reperfusion injury models, indicating the renal LEC response is both specific to where they lie in the lymphatic vasculature and the renal injury type. Conclusions: In this study, we uncover lymphatic vessel structural features of captured populations and injury-induced genetic changes. We further determine LEC gene expression is altered between injury models. How LECs respond to AKI may therefore be key in regulating future kidney disease progression. Overall design: Renal LECs were isolated from 5 male C57BL6/J mice treated with saline or cisplain (20mg/kg) 72 hours post injury. Renal LECs were isolated through dual magnetic enrichment of the markers CD31 and PDPN. A total of 10 kidneys were used per condition. Magnetic beads were cleaved after magnetic enrichment. LEC enrichment was confirmed by flow cytometry of CD45- Live Population expression of Cd31+, Pdpn+, and Lyve1+ marker presence.

背景：急性肾损伤（Acute Kidney Injury, AKI）引发的炎症反应很可能决定肾脏未来的健康状态。淋巴管通过物质转运与免疫调节功能维持组织稳态。由于肾脏内淋巴管内皮细胞（Lymphatic Endothelial Cells, LECs）相对稀少，过往的测序研究尚未对这类细胞及其对急性肾损伤的应答进行表征。
方法：本研究通过单细胞RNA测序（single-cell RNA sequencing）对小鼠肾脏淋巴管内皮细胞亚群进行表征，并探究其在顺铂诱导的急性肾损伤后72小时的变化。数据采用Seurat软件包进行处理。本研究通过从顺铂损伤及缺血再灌注损伤（ischemia reperfusion injury）模型中分离的淋巴管内皮细胞进行定量聚合酶链反应（quantitative Polymerase Chain Reaction, qPCR）验证研究结果，同时借助免疫荧光实验（immunofluorescence），并在体外培养的人淋巴管内皮细胞中确认了上述发现。
结果：本研究鉴定出了既往研究尚未表征的肾脏淋巴管内皮细胞及其淋巴管功能。本研究报道了对照组与顺铂损伤组之间独特的基因表达变化图谱。发生急性肾损伤后，肾脏淋巴管内皮细胞的基因表达发生改变，涉及内皮细胞凋亡、血管生成过程以及免疫调节信号通路与代谢活动。本研究还发现不同损伤模型之间存在差异：肾脏淋巴管内皮细胞在顺铂损伤与缺血再灌注损伤模型中的基因表达变化进一步存在不同，这表明肾脏淋巴管内皮细胞的应答既与其在淋巴管脉管系统中的位置相关，也取决于肾脏损伤的类型。
结论：本研究揭示了所捕获细胞群体的淋巴管结构特征以及损伤诱导的基因表达变化。本研究进一步证实，不同损伤模型之间淋巴管内皮细胞的基因表达存在差异。因此，淋巴管内皮细胞对急性肾损伤的应答机制，可能是调控肾脏疾病未来进展的关键环节。
整体实验方案：本研究从5只雄性C57BL/6J小鼠中分离肾脏淋巴管内皮细胞，这些小鼠分别经生理盐水或顺铂（20mg/kg）处理，于损伤后72小时取材。肾脏淋巴管内皮细胞通过针对标志物CD31与PDPN的双重磁珠富集法进行分离。每组共使用10个肾脏组织。磁珠富集完成后，磁珠被裂解脱离细胞。通过流式细胞术（flow cytometry）检测CD45-活细胞群体中Cd31+、Pdpn+及Lyve1+标志物的表达，确认了淋巴管内皮细胞的富集效果。